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Familial ALS with FUS P525L mutation: two Japanese sisters with multiple systems involvement
Journal of the Neurological Sciences 323 (2012) 85–92
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Familial ALS with FUS P525L mutation: two Japanese sisters with multiple systems involvement Yoko Mochizuki a,⁎, Eiji Isozaki b, Masaki Takao c, Tomoyo Hashimoto d, e, Makoto Shibuya a, f, Makoto Arai g, Masato Hosokawa g, Akihito Kawata b, Kiyomitsu Oyanagi d, e, Ban Mihara h, Toshio Mizutani a, i a
Department of Pathology, Tokyo Metropolitan Neurological Hospital, Tokyo 183–0042, Japan Department of Neurology, Tokyo Metropolitan Neurological Hospital, Tokyo 183–0042, Japan Division of Neuropathology, Mihara Memorial Hospital, Gunma 372–0006, Japan d Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183–8526, Japan e Division of Neuropathology, Department of Brain Disease Research, Shinshu University School of Medicine, Nagano, Japan f Department of Pathology, Tokyo Medical University Ibaraki Medical Center, Ibaraki 300–0395, Japan g Department of Neuropathology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156–8506, Japan h Department of Neurology, Mihara Memorial Hospital, Gunma 372–0006, Japan i Fuchu Medical Center for the Disabled, Tokyo, Japan b c
a r t i c l e
i n f o
Article history: Received 5 June 2012 Received in revised form 15 August 2012 Accepted 20 August 2012 Available online 11 September 2012 Keywords: Familial amyotrophic lateral sclerosis Basophilic inclusion Fused in sarcoma Juvenile amyotrophic lateral sclerosis Long survival Totally locked-in state Multiple systems involvement
a b s t r a c t We evaluated the clinicopathological features of familial amyotrophic lateral sclerosis (ALS) with the fused in sarcoma (FUS) P525L mutation. Two sisters and their mother had a similar clinical course, which was characterized by the development of limb weakness at a young age with rapid disease progression. An elder sister, patient 1, progressed into a totally locked-in state requiring mechanical ventilation and died 26 years after the onset of the disease. In contrast, the younger sister, patient 2, died in the early stages of the disease. The patients had neuropathological findings that indicated a very active degeneration of motor neurons and multiple system degeneration, which led to marked brain and spinal cord atrophy in the long term clinical outcome. The multiple system degeneration included the frontal lobe, the basal ganglia and substantia nigra, cerebellum and related area. Compared with previously reported ALS cases, the severe degeneration of the frontal lobe and the striatum were the characteristic features in the patient 1 in this case study. The degeneration spread over multiple systems might be caused not only by the appearance of the FUS immunoreactive neuronal cytoplasmic inclusions but also by the degeneration of neuronal connections from the primary motor cortex and related areas. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Sporadic amyotrophic lateral sclerosis (ALS) with basophilic cytoplasmic inclusions (BIs), characterized by an onset before 25 years of age with rapid progression, has been proposed to constitute a distinct clinical entity [1,2]. Bäumer et al. [3] demonstrated that the BIs in the cases of sporadic juvenile ALS were immunoreactive for fused in sarcoma (FUS), and two of their six patients had the FUS P525L mutation. Therefore, they proposed that juvenile ALS with BIs should be classified as ALS–FUS. Neuropathologically, Mackenzie et al. [4] divided patients with ALS–FUS in two groups: early-onset cases, including two with the FUS P525L mutation [3] and late-onset cases, including two with the FUS R521C mutation. Furthermore, they [4] suggested that patients with ALS–FUS and frontotemporal lobar degeneration (FTLD) with
⁎ Corresponding author at: Department of Pathology, Tokyo Metropolitan Neurological Hospital, 2-6-1 Musashidai Fuchu-shi, Tokyo 183–0042, Japan. Tel.: +82 423 23 5110. E-mail address: [email protected] (Y. Mochizuki). 0022-510X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2012.08.016
FUS-immunoreactive pathology (FTLD–FUS) were distinct because none of their ALS–FUS cases showed involvement of the broad range of neuroanatomical regions that occurs in the FTLD cases [5]. The neuropathological features of ALS with the FUS P525L mutation have been reported in only one other patient [6], who showed similar neuropathological features as to early-onset ALS–FUS [4]. This patient used non-invasive positive pressure ventilation during the last 4 months of her life; however, the other reported patients with the FUS P525L mutation [3,4] did not use respiratory assistance. Therefore, although it has been shown that an ALS case with the FUS R521C mutation, late-onset ALS–FUS showed no cerebral cortical involvement even in the prolonged stage with using mechanical ventilation [7], much is unknown about the clinicopathology of ALS with the FUS P525L mutation, early-onset ALS-FUS. Herein, we report the clinical and neuropathological findings in two autopsied sisters and their mother with Japanese familial ALS associated with the FUS P525L mutation. The two autopsied patients used mechanical ventilators: the elder sister used it for over 20 years; however, the younger sister only used it for approximately one year. Therefore, the difference between the clinicopathological
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findings of the elder sister at the late stage of the disease and those of the younger sister at the early stage of the disease are most likely indicative of the mechanism of disease progression. The significance of their neuroanatomical regions is discussed.
2. Case reports 2.1. Patient 1 The elder sister (proband, Subject III-1 in Fig. 1) exhibited a slight developmental delay in childhood. At the age of 13 years, she developed left and then right leg weakness, followed by quadriparesis. Her clinical feature was suggested to be a polyneuropathy. However, plasmapheresis was not effective for her. One year and 3 months after the onset, she was admitted to the Tokyo Metropolitan Neurological hospital because dysphagia, dyspnea, and dysarthria appeared. She was diagnosed as ALS and underwent a tracheostomy, with subsequent prolonged mechanical ventilation. Three years after the onset, she developed adduction paresis of the right eye, followed by ophthalmoparesis of both eyes. However, she was able to comprehend her situation. Head CT showed progressive brain atrophy (Fig. 2). Finally, all of her voluntary movement disappeared at the age of 23 years. Specifically, she progressed to a totally locked-in state that has been proposed [8] as one of the subgroups in the terminal condition of respirator-assisted long-survival ALS [9]. She died of pneumonia at the age of 40 years, 26 years after the onset.
2.3. Patient 2 Patient 2 (Subject III-2 in Fig. 1) was the younger sister of patient 1. She developed right hand weakness at the age of 25 years. One year after, she showed gait disturbance, dysphagia, and respiratory failure. Subsequently, she was placed on mechanical ventilation and was transferred to the Mihara Memorial hospital and evaluated by a neurologist. Neurological examination at the age of 26 years showed quadriplegia with hypotonia and decreased tendon reflexes in the all extremities, while Babinski's sign appeared. She died of pneumonia at the age of 27 years. 2.4. Patient 3 The mother of patients 1 and 2 (Subject II-4 in Fig. 1) had developed left arm weakness and died of progressive bulbar palsy within 6 months of the disease duration at the age of 35 years. 3. Methods 3.1. Neuropathological study The brain and spinal cord specimens were fixed with 20% buffered formalin and embedded in paraffin. Neuronal loss and/or fiber loss and gliosis was assessed in various regions of the nervous system using 10-μm-thick sections with hematoxylin and eosin (HE) and Klüver–Barrera (KB) stains. When necessary, Bodian, Nissl, periodic acid-Schiff (PAS) stain, luxol fast blue (LFB), cresyl violet, and Gallyas–Braak staining was performed.
2.2. Genetic analysis 3.2. Immunohistochemistry for BIs In patient 1, DNA was extracted from the patient's leukocytes using a conventional method with informed consent. All of the coding regions and exon–intron boundaries of the FUS gene were examined by direct sequencing of polymerase chain reaction (PCR) products. Detailed information regarding the PCR amplification conditions is available from the authors upon request. Sequencing of the PCR products was performed using a BigDye Terminator Cycle Sequencing Reaction kit (Life Technologies Japan) and an ABI PRISM 3100 Genetic Analyzer (Life Technologies Japan). The sequence analysis of the FUS gene identified a proline 525 to leucine (P525L) mutation. Therefore, we performed a deep resequencing analysis of the target gene and confirmed the presence of a rare heterozygous C-to-T transition at cDNA position 1574, resulting in a P525L missense mutation within the arginine-glycine-glycine motif of exon 15.
For the immunohistochemistry, 6-μm-thick sections were prepared. Specimens of the frontal lobe, hippocampus with medial temporal lobe, and pons were immunostained for ubiquitin (DAKO, 1:600), 43-kDa TAR DNA binding protein-43 (TDP-43) (Polyclonal Protein Tech Group, 1:500), α-internexin (Cosmo Bio, 1:250), α-synuclein (Santa Cruz Biotechnology, 1:400), and phosphorylation-dependent τ (AT8; Innogenetics, 1:5000), and the specimens of each cerebral lobe, basal ganglia, cerebellum, brainstem, and spinal cord were immunostained for FUS (Sigma, 1: 100) using a labeled streptavidin–biotin method. 3.3. Electron microscopical study of BIs Several pieces of formalin-fixed inferior frontal cortex and pontine nuclei of patient 1 were postfixed with 4% osmium tetroxide and conventionally processed for electron microscopy (Hitachi H-9000). 4. Results 4.1. Neuropathological findings in patient 1
Fig. 1. Pedigree of the family. The arrow indicates the proband. The affected individuals are represented by the solid black symbols. I-4 lived to be more than 81 years old.
The brain weighed 715 g. Marked cerebral atrophy was observed in the brainstem and cerebellar regions (Fig. 3A). The frontal white matter was marked with atrophy, the caudate nucleus was thin, and the putamen and globus pallidus were atrophic and brownish in color (Fig. 3B). The ventral lateral nucleus of the thalamus showed severe atrophy. However, the limbic system, including the medial temporal area, mammillary body, and cingulate gyrus were preserved. The brainstem and spinal cord were markedly atrophic (Fig. 4A), and the anterior horn of the spinal cord (Fig. 4B) and all motor nuclei of the brainstem showed severe neuron loss and gliosis (Table 1). Although some neurons were observed in the intermediolateral nucleus, neurons in Clarke's nucleus were markedly decreased. The dorsal root ganglion cells were preserved. In the spinal cord, although the posterior column was preserved, almost all of the fibers from the other areas were lost
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Fig. 2. Head CT of patient 1. (A) Mild frontal atrophy was observed at 2 years after the onset. (B) Moderate cerebral atrophy, particularly in the frontal lobe, and mild atrophy of the caudate nucleus were observed at 6 years after the onset.
Fig. 3. Macroscopic findings and histology in patient 1. (A) Marked atrophy of the brain are observed, especially in the frontal lobe. (B) In the coronal section, enlargement of the anterior horn of the lateral ventricle, and atrophy of the striatum and frontal lobe with thinner corpus callosum are observed; however, the amygdala and temporal lobe are preserved. (A, B: bar=1 cm). (C) Primary motor cortex showing neuronal loss with astroglial proliferation and spongiform changes in the upper cortical layers. (Bar=500 μm). (D) Caudate nucleus showing marked neuronal loss with gliosis. The arrow indicates a basophilic cytoplasmic inclusion. (Bar=50 μm, C, D: hematoxylin and eosin staining).
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Fig. 4. Cervical spinal cord features. (A) Marked atrophy is observed at C7. The posterior column is relatively preserved while the numbers of other fibers were decreased. (A–C: patient 1) (B) Marked neuronal loss and gliosis of the anterior horn are observed at C7. (C) Marked fiber loss in both the lateral corticospinal tract and spinocerebellar tract (*) is observed at C7. (D) Degeneration in the anterior horn and lateral to the anterior column, particularly in pyramidal tract and preserved posterior column, are observed in the cervical spinal cord. (D–F: patient 2) (E) Marked neuronal loss and gliosis of the cervical anterior horn is observed. (F) Many macrophages and astroglial proliferation are observed in the lateral corticospinal tract. (A, D, F: Klüver–Barrera staining, bar = 0.5 cm, B, E: hematoxylin and eosin staining, C: Bodian staining, B, C, E, F: bar = 50 μm).
(Fig. 4A, C). Nerve fibers of the brainstem were barely observed except for relative preservation in the temporoparietooccipitopontine tract, medial lemniscus, pontocerebellar fibers and middle cerebellar peduncle. Only a few Betz cells were observed and these were atrophic and generalized astroglial proliferation in all layers were observed in the primary motor cortex. Furthermore, the primary motor cortex, primary somatosensory cortex, and frontal and parietal cortices showed neuronal loss with gliosis. In these cortices, the upper cortical layers showed spongiform changes, and the deep cortical layers showed a loss of the radiating myelinated fibers (Fig. 3C). Nerve fibers in the frontal white matter were markedly decreased in number. The following systems were severely affected by the disease (Table 1): the basal ganglia, which were severely affected in the striatum (Fig. 3D), subthalamic nucleus, and substantia nigra, and were mildly affected in the globus pallidus; the cerebellar dentate nucleus and efferent fibers and the red nucleus; the pontine nucleus, the inferior olive, and Purkinje cells. The presence of BIs were difficult to establish in the lower motor neurons, the neurons of the substantia nigra and in the cerebellar dentate nucleus because of their marked neuronal loss (Table 1). The caudate nucleus showed severe degeneration, and many BIs were found there (Fig. 3D). In contrast, BIs were rarely found in the
limbic area and occipital cortex. The BIs were round in shape and compactly or loosely packed (Fig. 5A), sometimes with distinct basophilic rims. The BIs were positive with Bodian staining, while negative with PAS, cresyl violet, LFB, or Gallya–Braak staining.
4.2. Neuropathological findings in patient 2 Limited anatomical regions were available for neuropathological analysis. The spinal cord atrophy was mild (Fig. 4D), and the anterior horn and hypoglossal nucleus showed mild atrophy in spite of marked neuronal loss with astroglial proliferation (Fig. 4E). Nerve fiber loss with numerous macrophages was observed in the corticospinal tract (Fig. 4F) and spinocerebellar tract. The corticospinal tract showed the same features as the internal capsule. However, the primary motor cortex, the hippocampus and the amygdala were not examined. Numerous BIs were observed in extensive areas with slight degeneration (Table 1). The shape of the BIs in patient 2 was similar to that in patient 1, especially in the anterior horn of the spinal cord and the pigmented neurons of the substantia nigra. However, some BIs in the globus pallidus, thalamus, pontine nucleus, inferior olivary nucleus, and dentate nucleus of
Y. Mochizuki et al. / Journal of the Neurological Sciences 323 (2012) 85–92 Table 1 Neuropathological findings.
Motor neurons Primary motor cortex Anterior horn of the spinal cord Hypoglossal nucleus Oculomotor nucleus Frontal cortex Basal ganglia and substantia nigra Caudate nucleus Putamen Globus pallidus Substantia nigra Cerebellum and related area Purkinje cells Cerebellar dentate nucleus Red nucleus Inferior olivary nucleus Pontine nucleus
Patient
1
Patient
2
DEG
BI
NCI
DEG
BI
NCI
++ +++ +++ +++ ++
2 1 − 1 2
2 1 − 1 2
n +++ ++ − +
n 2 1 1 1
n 3 1 1 1
+++ +++ ++ +++
3 2 1 −
3 1 − −
+ − + +
1 1 1 1
2 2 3 3
++ +++ ++ ++ ++
− 1 − − 2
− − − 1 2
+ + + − −
− 2 1 2 1
− 3 1 3 2
DEG: degeneration assessed on the hematoxylin and eosin-, Klüver-Barrera-stained sections. The degeneration was indicated as absent (−), slight (+), mild (++) or severe (+++). BIs: basophilic inclusions. NCI: FUS-immunoreactive (ir) neuronal cytoplasmic inclusion, n: not evaluated (or not examined) The BIs and the FUS pathology was indicated as none (−), rare (1), occasional (2), frequently (3).
patient 2 were irregular in shape. Moreover, in patient 2, some cytoplasmic inclusions showed weak eosinophilia. 4.3. Immunohistochemistry of BIs Both the BIs and the weak eosinophilic inclusions were strongly immunoreactive (ir) for FUS (Fig. 5B). The FUS-ir neuronal cytoplasmic inclusions (NCIs) were more numerous and widespread than NCIs identified with HE, KB, or Bodian staining (Table 1). A few FUS-ir neuronal intranuclear small round inclusions were observed in the cerebral cortex in patient 1 (Fig. 5C) and in the cerebellar dentate nucleus in patient 2. Some FUS-ir granular deposits were observed in the neuronal cytoplasm and neuritis (Fig. 5D). The BIs were slightly positive or negative for ubiquitin, while negative for TDP-43, α-internexin, AT8, and α-synuclein immunoreactivity. Some FUS-ir glial inclusions were observed in the cerebral cortex and the white matter in patient 1. The FUS-ir NCIs of the cerebral cortex in patient 1 appeared mainly in the frontal cortex, including the primary motor cortex and, to some extent, in the superior parietal lobe but rarely in the primary somatosensory cortex, temporal cortex or occipital cortex. There was no immunoreactivity in the granular neurons, while a few pyramidal neurons of the hippocampus were immunoreactive for FUS. 4.4. Electron microscopical findings of BIs The BIs in patient 1 were loose clusters of filamentous structures associated with granules and had no limiting membrane (Fig. 5E). The BIs occasionally contained cytoplasmic organelles, such as mitochondria, but no neurofilaments with side arms or twisted tubules were found in them (Fig. 5F). 5. Discussion All three patients in the family described in this case study developed limb weakness followed by bulbar palsy at a young age with a very rapid progression. In patient 1, the FUS P525L mutation was identified. Therefore, although a gene analysis was not conducted in the other two affected patients, it seems likely that they had the same FUS mutation. This clinical phenotype is similar to that of the already reported juvenile ALS patients (Table 2) [1–4,6,10–16]. Furthermore, similar to patient 1, some reported juvenile ALS patients
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had developmental delays [1,3,6,14], which suggests an early manifestation of the disease. Despite the short disease duration in patient 2 and the juvenile ALS patients (Table 2) [1–4,6,12–16], their lower motor neuron degeneration was severe (Fig. 4D, E), which seems to be reflected in their very rapid progression of paralysis. Without obvious symptoms of upper motor neuron impairment, the corticospinal tract of patient 2 and patient 8 [6] in Table 2 showed very active mobilization of macrophages (Fig. 4F). These findings suggest that the lower motor neuron degeneration developed earlier than the upper motor neuron degeneration, and both of them were very active. Therefore, in patient 1, it is probable that the very severe degeneration of both the lower and upper motor neurons (Fig. 4A–C), including the primary motor cortex (Fig. 3C) came from those very active features in the early stages of the disease. Furthermore, patient 1 showed severe involvement of multiple systems (Table 1). In all of the regions of patient 2, neuronal loss and gliosis were slight, whereas frequent BIs/FUS-ir NCIs appeared (Table 1). In contrast, the limbic system was preserved with rare BIs/FUS-ir NCIs. These findings suggest that the neuronal loss was preceded by the appearance of the BIs/FUS-ir NCIs. In the juvenile ALS patients (Table 2) [1–4,12,14], although various regions besides motor neurons had the BIs/FUS-ir NCIs, these regions did not showed neuronal loss. The disease duration of the juvenile ALS patients was shorter than patients of this case study because the patients of this case study used mechanical ventilation for more than one year. These results might indicate that the initiation of a neuronal loss needs some period of time from the appearance of the BIs/FUS-ir NCIs to occur. Considering the neuroanatomical lesions, the marked fiber loss in the brainstem and spinal cord (Fig. 4A) of patient 1 appeared similar to respirator-assisted long-survival ALS patients, who progressed into a totally locked-in state [8,17–20]. However, their neuroanatomical lesions are various. Although all the patients in these previous studies [8,17–20] showed severe degeneration of the globus pallidus, substantia nigra, and subthalamic nucleus, degeneration of the striatum and the brain atrophy were not present or were mild [8,17–19], except in one patient [20]. This last patient showed the marked frontal lobe atrophy and the multiple system degeneration including the striatum that were also observed in patient 1. However, the patient from the previous study had widespread TDP-43-ir NCI [20]. Therefore, it was suggested that the degeneration of the striatum was correlated with the frontal atrophy of either FUS or TDP-43 pathology. In view of the connections among neurons, the striatum is well known to be the main entry point from the motor-related cortex and connected by the radiating fibers, which come from the deep cortical layers. The loss of radiating fibers in patient 1 corresponded to this feature. The other degenerated lesions in the pontine nucleus and subthalamic nucleus were also connected with the motor-related cortex. Furthermore, in the upper cortical layer, degeneration was contributed to loss of the association fibers, which connected the cerebral cortex. Therefore, the loss of the radiating and associated fibers in the primary motor cortex and the related cortex might induce multiple system involvement, concomitantly with the cerebral white matter atrophy. Regarding the respirator-assisted long-survival ALS with the other mutation in the FUS gene, there has been one ALS patient with the FUS R521C mutation. This patient became quadriplegic with ophthalmoparesis and neuropathologically showed multiple systems degeneration with the BIs/FUS-ir NCI and FUS-ir glial cytoplasmic inclusions [7]. Among the findings of the present case study, the feature of ganglion cell loss of the dorsal root ganglia posterior column degeneration was not observed. Another study has described a patient suggested to have the FUS R521C mutation, with similar findings [21]. Her disease duration was only three years without using mechanical ventilation. Therefore, posterior column degeneration is considered to be a characteristic of ALS with the
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Fig. 5. Basophilic cytoplasmic inclusions (BIs). (A) BIs (arrows) are round (hematoxylin and eosin staining). (B) BIs are strongly immunoreactive (ir) for fused in sarcoma (FUS). (A, B: primary motor cortex of patient 1). (C) FUS-ir neuronal intranuclear inclusions are small and round. (D) FUS-ir deposits are rarely found in neurites. (C, D: parietal cortex of patient 1. A–D: bar = 10 μm). (E) Electron micrograph of a BI (arrows). Note its globular shape with no limiting membrane. (Pons of patient 1, N: nucleus, *BI, bar = 5 μm). (F) At higher magnification, the filamentous structures appeared to be straight or curved with a diameter of range from 15 to 25 nm and had no side arm or constriction. The granules were 20–30 nm in diameter. (Inferior frontal cortex of patient 1, bar = 500 nm, C, D: uranyl acetate and lead citrate staining.)
FUS R521C mutation. According to this feature, it is probable that there are some differences in the disease phenotype between ALS patients with the FUS P525L mutations and those with R521C mutations. Furthermore, the findings of ALS patients with the FUS R521C mutations are of interest because posterior column degeneration is known to be the characteristic of ALS patients with a mutation of the copper/zinc superoxide dismutase (SOD1) gene. A patient with the SOD1 V118L mutation, who progressed into a totally locked-in state, showed posterior column degeneration [19]. Based on the finding that posterior column degeneration is characteristic in ALS patients with either FUS or SOD1 gene mutations, the disease phenotype may have some factors besides gene mutation. Although variability in the morphology and tinctorial characteristics of BIs has been noted [4], the ultrastructural profiles of BIs in the present patients were essentially similar to those of BIs in other diseases that were recently reported to have a FUS-ir pathology [22]. The diseases with BIs and a FUS-ir pathology include juvenile sporadic ALS [2,6,14], adult-onset sporadic ALS [23,24], and generalized
variants of Pick's disease [25], which are now classified as basophilic inclusion body disease subtypes of FTLD-FUS [5,26]. Furthermore, the findings of striatum and frontal lobe atrophy with frequent FUS-ir NCIs [5,22–29] were the same as the findings observed in the present patients. However, the preserved amygdala and hippocampus and no vermiform FUS-ir neuronal intranuclear inclusions, a consistent feature of FTLD-FUS observed in the present patients were different from the findings of FTLD-FUS patients [5,26]. Therefore, ALS with the FUS P525L mutation might have a partially common pathology to FTLD-FUS. In conclusion, we examined three members in a family with a very rapid progressive ALS with the FUS P525L mutation. In contrast to patient 1, who progressed into a totally locked-in state and showed multiple systems degeneration in addition to marked motor neuron degeneration, patient 2 died in the early stage of the disease and showed very active motor neuron degeneration and slight multiple systems degeneration with frequent BIs/FUS-ir NCIs. The clinicopathological findings of these patients indicate that the degeneration
− UE −
− + − − − − [15]
+ UE −
− + − − − − [14]
− UE Dementia, ophthalmoparesis, autonomic symptom
Conflict of interest + Various locations + − − − − [13] [14] + + − + + + [1] + + − + NR NR [3,4]
Acknowledgments
+ + − − − − [6] + + − − − + [3] W: woman, M: man, NIPPV: non−invasive positive pressure ventilation, NR: not recorded, LE: lower exteremities, UE: upper extremities. # Deletion mutation in exon 15 of the FUS gene.
+ + + + + + [2] + + − − − − [6] + + + + + + [3,4] Apearance of FUS-immunoreactive neuronal cytoplasmic inclusions or basophilic inclusions Motor cortex + NR No No No autopsy Lower motor neurons + + autopsy autopsy Basal ganglia + + Pontine nucleus + + Cerebellar dentate nucleus + + Substantia nigra − + Reference Present patients [10] [11]
+ + − + + + [3,4]
+ − UE UE − − − UE − − Hip − NR NR NR − UE NR + NR LE UE Ophthalmoparesis −
The authors have no conflicts of interest to report.
+ + − − − + [12]
+ − UE UE − Autonomic symptom + UE −
No 24 W 7 No 16 W 12
No 25 W 37 * 27 − UE −
No 21 W 4
− NR −
No 12 W 12 No 18 W 6
No 13 W 20 * 4 (NIPPV) + LE −
No 15 M 18
Sporadic juvenile ALS with basophilic cytoplasmic inclusions #
No 18 W 11 No 22 W 10
Yes 16–32 2 W, 2 M b12 in 3, b24 in 1 − UE, Bulbar − Yes 22 NR 6 Yes 35 W 6 Yes 27 W 28 * 14 FUS P525L mutation
Yes 13 W 312 *294
Famiy history Age at onset (years) Gender Duration*respiratory assist (months) Developmental delay Initial symptom Atypical symptoms as ALS
91
spread over multiple systems during ALS with the FUS P525L mutation is caused by not only the appearance of the BIs/FUS-ir NCIs but also the degeneration of the neuronal connection from the primary motor cortex and related areas. The degeneration of the striatum might be considered to be the cause of frontal lobe degeneration in ALS patients.
− + − − − − [16]
No 11 W 12 No 22 W 6 No 19 W 32 *several
− UE −
18 6 5 4 3 2 1 Patient
Table 2 Clinical features and distribution of basophilic cytoplasmic inclusions of juvenile amyotrophic lateral sclerosis.
7
8
9
10
11
12
13
14
15
16
17
19
No 14 M b12
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The authors are grateful to J. Motoki, A. Ishihara, Y. Sato, and others in the laboratory at the Tokyo Metropolitan Neurological Hospital and to K. Suwabe, S. Aoyagi, M. Tano, and E. Kawakami at the Tokyo Metropolitan Institute for Neuroscience for their excellent technical assistance. This study was supported in part by grants from the Japan Society for the Promotion of Science (JSPS) Kakenhi Grant-in-Aid for Scientific Research (B) 22390429 and a grant from the Joint Program for ALS Research, Tokyo Metropolitan Institute of Medical Science (Y. M), and Research on Measures for Intractable Diseases (H23-nanchi-ippan-06 and H23-nanchi-ippan-013), a Grant-in-Aid for Scientific Research (C, 23500435) and a Grantin-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the Ministry of Education, Culture, Sports, Science and Technology (M. T.), and the Japanese Ministry of Education, Science, Sports and Culture, Basic Research (B) #22300177 (K. O.). References [1] Nelson JS, Prensky AL. Sporadic juvenile amyotrophic lateral sclerosis. A clinicopathological study of a case with neuronal cytoplasmic inclusions containing RNA. Arch Neurol 1972;27:300-6. [2] Oda M, Akagawa N, Tabuchi Y, Tanabe H. A sporadic juvenile case of the amyotrophic lateral sclerosis with neuronal intracytoplasmic inclusions. Acta Neuropathol 1978;44:211-6. [3] Bäumer D, Hilton D, Paine SML, Turner MR, Lowe J, Talbot K, et al. Juvenile ALS with basophilic inclusions is a FUS proteinopathy with FUS mutations. Neurology 2010;75:611-8. [4] Mackenzie IRA, Ansorge O, Strong M, Bilbao J, Zinman L, Ang LC, et al. Pathological heterogeneity in amyotrophic lateral sclerosis with FUS mutations: two distinct patterns correlating with disease severity and mutation. Acta Neuropathol 2011;122:87-98. [5] Mackenzie IRA, Munoz DG, Kusaka H, Yokota O, Ishihara K, Roeber S, et al. Distinct pathological subtypes of FTLD-FUS. Acta Neuropathol 2011;121:207-18. [6] Huang EJ, Zhang J, Geser F, Trojanowski JQ, Strober JB, Dickson DW, et al. Extensive FUS-immunoreactive pathology in juvenile amyotrophic lateral sclerosis with basophilic inclusions. Brain Pathol 2010;20:1069-76. [7] Tateishi T, Hokonohara T, Yamasaki R, Miura S, Kikuchi H, Iwaki A, et al. Multiple system degeneration with basophilic inclusions in Japanese ALS patients with FUS mutation. Acta Neuropathol 2010;119:355-64. [8] Hayashi H, Kato S. Total manifestations of amyotrophic lateral sclerosis. ALS in the totally locked-in state. J Neurol Sci 1989;93:19-35. [9] Hayashi H, Oppenheimer EA. ALS patients on TPPV: totally locked-in state,159 neurologic findings and ethical implications. Neurology 2003;61:135-7. [10] Kwiatkowski Jr TJ, Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 2009;323:1205-8. [11] Chiò A, Restagno G, Brunetti M, Ossola I, Calvo A, Mora G, et al. Two Italian kindreds with familial amyotrophic lateral sclerosis due to FUS mutation. Neurobiol Aging 2009;30:1272-5. [12] Aizawa H, Kimura T, Hashimoto K, Yahara O, Okamoto K, Kikuchi K. Basophilic cytoplasmic inclusions in a case of sporadic juvenile amyotrophic lateral sclerosis. J Neurol Sci 2000;176:109-13. [13] Berry RG, Chambers RA, Duckett S. Clinicopathological study of juvenile ALS. Neurology 1969;19:312. [14] Matsumoto S, Kusaka H, Murakami N, Hashizume Y, Okazaki H, Hirano A. Basophilic inclusions in sporadic juvenile amyotrophic lateral sclerosis: an immunocytochemical and ultrastructural study. Acta Neuropathol 1992;83:579-83. [15] Tsujihata M, Taguchi H, Oku Y, Takamori M, Terao H. A sporadic case of juvenile ALS. Rinsho Shinkeigaku 1978;18:82-8 (in Japanese with English abstract). [16] Wohlfart G, Swank RL. Pathology of ALS. Fiber analysis of the ventral roots and pyramidal tracts of the spinal cord. Arch Neurol Psychiatry 1941;46:783-99.
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[17] Mizutani T, Sakamaki Y, Tsuchiya N, Kamei S, Kohzu H, Horiuchi R, et al. Amyotrophic lateral sclerosis with ophthalmoplegia and multisystem degeneration in patients on long-term use of respirator. Acta Neuropathol 1992;84:372-7. [18] Kato S, Oda M, Hayashi H. Neuropathology in amyotrophic lateral sclerosis patients on respirators: uniformity and diversity in 13 cases. Neuropathology 1993;13:229-36. [19] Shimizu T, Kawata A, Kato S, Hayashi M, Takamoto K, Hayashi H, et al. Autonomic failure in ALS with a novel SOD1 gene mutation. Neurology 2000;54:1534-7. [20] Nishihara Y, Tan C-F, Toyoshima Y, Yonemochi Y, Kondo H, Nakajima T, et al. Sporadic amyotrophic lateral sclerosis: widespread multisystem degeneration with TDP-43 pathology in a patient after long-term survival on a respirator. Neuropathology 2009;29:689-96. [21] Kobayashi Z, Tsuchiya K, Arai T, Aoki M, Hasegawa M, Ishizu H, et al. Occurrence of basophilic inclusions and FUS-immunoreactive neuronal and glial inclusions in a case of familial amyotrophic lateral sclerosis. J Neurol Sci 2010;29:6–11. [22] Munoz DG, Neumann M, Kusaka H, Yokota O, Ishihara K, Terada S, et al. FUS pathology in basophilic inclusion body disease. Acta Neuropathol 2009;118: 617-27. [23] Kusaka H, Matsumoto S, Imai T. An adult-onset case of sporadic motor neuron disease with basophilic inclusions. Acta Neuropathol 1990;80:660-85.
[24] Kusaka H, Matsumoto S, Imai T. Adult-onset motor neuron disease with basophilic intraneuronal inclusion bodies. Clin Neuropathol 1993;12:215-8. [25] Munoz-Garcia D, Ludwin SK. Classic and generalized variants of Pick's disease: a clinicopathological, ultrastructural and immunocytochemical comparative study. Ann Neurol 1984;16:467-80. [26] Neumann M, Rademakers R, Roeber S, Baker M, Kretzcjmar HA, Mackenzie IRA. A new subtype of frontotemporal lobar degeneration with FUS pathology. Brain 2009;132:2922-31. [27] Ito H, Kusaka H, Matsumoto S, Imai T. Topographic involvement of the striatal efferents in basal ganglia of patients with adult-onset motor neuron disease with basophilic inclusions. Acta Neuropathol 1995;89:513-8. [28] Seelaar H, Klijnsma KY, de Koning I, van der Lught A, Chiu WZ, Azmani A, et al. Frequency of ubiquitin and FUS-positive, TDP-43-negative frontotemporal lobar degeneration. J Neurol 2010;257:747-53. [29] Snowden JS, Hu Q, Rollinson S, Halliwell N, Robinson A, Davidson YS, et al. The most common type of FTLD-FUS (aFTLD-U) is associated with a distinct clinical form of frontotemporal dementia but is not related to mutations in the FUS gene. Acta Neuropathol 2011;122:99–110.
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Familial ALS with FUS P525L mutation: two Japanese sisters with multiple systems involvement Yoko Mochizuki a,⁎, Eiji Isozaki b, Masaki Takao c, Tomoyo Hashimoto d, e, Makoto Shibuya a, f, Makoto Arai g, Masato Hosokawa g, Akihito Kawata b, Kiyomitsu Oyanagi d, e, Ban Mihara h, Toshio Mizutani a, i a
Department of Pathology, Tokyo Metropolitan Neurological Hospital, Tokyo 183–0042, Japan Department of Neurology, Tokyo Metropolitan Neurological Hospital, Tokyo 183–0042, Japan Division of Neuropathology, Mihara Memorial Hospital, Gunma 372–0006, Japan d Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183–8526, Japan e Division of Neuropathology, Department of Brain Disease Research, Shinshu University School of Medicine, Nagano, Japan f Department of Pathology, Tokyo Medical University Ibaraki Medical Center, Ibaraki 300–0395, Japan g Department of Neuropathology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156–8506, Japan h Department of Neurology, Mihara Memorial Hospital, Gunma 372–0006, Japan i Fuchu Medical Center for the Disabled, Tokyo, Japan b c
a r t i c l e
i n f o
Article history: Received 5 June 2012 Received in revised form 15 August 2012 Accepted 20 August 2012 Available online 11 September 2012 Keywords: Familial amyotrophic lateral sclerosis Basophilic inclusion Fused in sarcoma Juvenile amyotrophic lateral sclerosis Long survival Totally locked-in state Multiple systems involvement
a b s t r a c t We evaluated the clinicopathological features of familial amyotrophic lateral sclerosis (ALS) with the fused in sarcoma (FUS) P525L mutation. Two sisters and their mother had a similar clinical course, which was characterized by the development of limb weakness at a young age with rapid disease progression. An elder sister, patient 1, progressed into a totally locked-in state requiring mechanical ventilation and died 26 years after the onset of the disease. In contrast, the younger sister, patient 2, died in the early stages of the disease. The patients had neuropathological findings that indicated a very active degeneration of motor neurons and multiple system degeneration, which led to marked brain and spinal cord atrophy in the long term clinical outcome. The multiple system degeneration included the frontal lobe, the basal ganglia and substantia nigra, cerebellum and related area. Compared with previously reported ALS cases, the severe degeneration of the frontal lobe and the striatum were the characteristic features in the patient 1 in this case study. The degeneration spread over multiple systems might be caused not only by the appearance of the FUS immunoreactive neuronal cytoplasmic inclusions but also by the degeneration of neuronal connections from the primary motor cortex and related areas. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Sporadic amyotrophic lateral sclerosis (ALS) with basophilic cytoplasmic inclusions (BIs), characterized by an onset before 25 years of age with rapid progression, has been proposed to constitute a distinct clinical entity [1,2]. Bäumer et al. [3] demonstrated that the BIs in the cases of sporadic juvenile ALS were immunoreactive for fused in sarcoma (FUS), and two of their six patients had the FUS P525L mutation. Therefore, they proposed that juvenile ALS with BIs should be classified as ALS–FUS. Neuropathologically, Mackenzie et al. [4] divided patients with ALS–FUS in two groups: early-onset cases, including two with the FUS P525L mutation [3] and late-onset cases, including two with the FUS R521C mutation. Furthermore, they [4] suggested that patients with ALS–FUS and frontotemporal lobar degeneration (FTLD) with
⁎ Corresponding author at: Department of Pathology, Tokyo Metropolitan Neurological Hospital, 2-6-1 Musashidai Fuchu-shi, Tokyo 183–0042, Japan. Tel.: +82 423 23 5110. E-mail address: [email protected] (Y. Mochizuki). 0022-510X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2012.08.016
FUS-immunoreactive pathology (FTLD–FUS) were distinct because none of their ALS–FUS cases showed involvement of the broad range of neuroanatomical regions that occurs in the FTLD cases [5]. The neuropathological features of ALS with the FUS P525L mutation have been reported in only one other patient [6], who showed similar neuropathological features as to early-onset ALS–FUS [4]. This patient used non-invasive positive pressure ventilation during the last 4 months of her life; however, the other reported patients with the FUS P525L mutation [3,4] did not use respiratory assistance. Therefore, although it has been shown that an ALS case with the FUS R521C mutation, late-onset ALS–FUS showed no cerebral cortical involvement even in the prolonged stage with using mechanical ventilation [7], much is unknown about the clinicopathology of ALS with the FUS P525L mutation, early-onset ALS-FUS. Herein, we report the clinical and neuropathological findings in two autopsied sisters and their mother with Japanese familial ALS associated with the FUS P525L mutation. The two autopsied patients used mechanical ventilators: the elder sister used it for over 20 years; however, the younger sister only used it for approximately one year. Therefore, the difference between the clinicopathological
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findings of the elder sister at the late stage of the disease and those of the younger sister at the early stage of the disease are most likely indicative of the mechanism of disease progression. The significance of their neuroanatomical regions is discussed.
2. Case reports 2.1. Patient 1 The elder sister (proband, Subject III-1 in Fig. 1) exhibited a slight developmental delay in childhood. At the age of 13 years, she developed left and then right leg weakness, followed by quadriparesis. Her clinical feature was suggested to be a polyneuropathy. However, plasmapheresis was not effective for her. One year and 3 months after the onset, she was admitted to the Tokyo Metropolitan Neurological hospital because dysphagia, dyspnea, and dysarthria appeared. She was diagnosed as ALS and underwent a tracheostomy, with subsequent prolonged mechanical ventilation. Three years after the onset, she developed adduction paresis of the right eye, followed by ophthalmoparesis of both eyes. However, she was able to comprehend her situation. Head CT showed progressive brain atrophy (Fig. 2). Finally, all of her voluntary movement disappeared at the age of 23 years. Specifically, she progressed to a totally locked-in state that has been proposed [8] as one of the subgroups in the terminal condition of respirator-assisted long-survival ALS [9]. She died of pneumonia at the age of 40 years, 26 years after the onset.
2.3. Patient 2 Patient 2 (Subject III-2 in Fig. 1) was the younger sister of patient 1. She developed right hand weakness at the age of 25 years. One year after, she showed gait disturbance, dysphagia, and respiratory failure. Subsequently, she was placed on mechanical ventilation and was transferred to the Mihara Memorial hospital and evaluated by a neurologist. Neurological examination at the age of 26 years showed quadriplegia with hypotonia and decreased tendon reflexes in the all extremities, while Babinski's sign appeared. She died of pneumonia at the age of 27 years. 2.4. Patient 3 The mother of patients 1 and 2 (Subject II-4 in Fig. 1) had developed left arm weakness and died of progressive bulbar palsy within 6 months of the disease duration at the age of 35 years. 3. Methods 3.1. Neuropathological study The brain and spinal cord specimens were fixed with 20% buffered formalin and embedded in paraffin. Neuronal loss and/or fiber loss and gliosis was assessed in various regions of the nervous system using 10-μm-thick sections with hematoxylin and eosin (HE) and Klüver–Barrera (KB) stains. When necessary, Bodian, Nissl, periodic acid-Schiff (PAS) stain, luxol fast blue (LFB), cresyl violet, and Gallyas–Braak staining was performed.
2.2. Genetic analysis 3.2. Immunohistochemistry for BIs In patient 1, DNA was extracted from the patient's leukocytes using a conventional method with informed consent. All of the coding regions and exon–intron boundaries of the FUS gene were examined by direct sequencing of polymerase chain reaction (PCR) products. Detailed information regarding the PCR amplification conditions is available from the authors upon request. Sequencing of the PCR products was performed using a BigDye Terminator Cycle Sequencing Reaction kit (Life Technologies Japan) and an ABI PRISM 3100 Genetic Analyzer (Life Technologies Japan). The sequence analysis of the FUS gene identified a proline 525 to leucine (P525L) mutation. Therefore, we performed a deep resequencing analysis of the target gene and confirmed the presence of a rare heterozygous C-to-T transition at cDNA position 1574, resulting in a P525L missense mutation within the arginine-glycine-glycine motif of exon 15.
For the immunohistochemistry, 6-μm-thick sections were prepared. Specimens of the frontal lobe, hippocampus with medial temporal lobe, and pons were immunostained for ubiquitin (DAKO, 1:600), 43-kDa TAR DNA binding protein-43 (TDP-43) (Polyclonal Protein Tech Group, 1:500), α-internexin (Cosmo Bio, 1:250), α-synuclein (Santa Cruz Biotechnology, 1:400), and phosphorylation-dependent τ (AT8; Innogenetics, 1:5000), and the specimens of each cerebral lobe, basal ganglia, cerebellum, brainstem, and spinal cord were immunostained for FUS (Sigma, 1: 100) using a labeled streptavidin–biotin method. 3.3. Electron microscopical study of BIs Several pieces of formalin-fixed inferior frontal cortex and pontine nuclei of patient 1 were postfixed with 4% osmium tetroxide and conventionally processed for electron microscopy (Hitachi H-9000). 4. Results 4.1. Neuropathological findings in patient 1
Fig. 1. Pedigree of the family. The arrow indicates the proband. The affected individuals are represented by the solid black symbols. I-4 lived to be more than 81 years old.
The brain weighed 715 g. Marked cerebral atrophy was observed in the brainstem and cerebellar regions (Fig. 3A). The frontal white matter was marked with atrophy, the caudate nucleus was thin, and the putamen and globus pallidus were atrophic and brownish in color (Fig. 3B). The ventral lateral nucleus of the thalamus showed severe atrophy. However, the limbic system, including the medial temporal area, mammillary body, and cingulate gyrus were preserved. The brainstem and spinal cord were markedly atrophic (Fig. 4A), and the anterior horn of the spinal cord (Fig. 4B) and all motor nuclei of the brainstem showed severe neuron loss and gliosis (Table 1). Although some neurons were observed in the intermediolateral nucleus, neurons in Clarke's nucleus were markedly decreased. The dorsal root ganglion cells were preserved. In the spinal cord, although the posterior column was preserved, almost all of the fibers from the other areas were lost
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Fig. 2. Head CT of patient 1. (A) Mild frontal atrophy was observed at 2 years after the onset. (B) Moderate cerebral atrophy, particularly in the frontal lobe, and mild atrophy of the caudate nucleus were observed at 6 years after the onset.
Fig. 3. Macroscopic findings and histology in patient 1. (A) Marked atrophy of the brain are observed, especially in the frontal lobe. (B) In the coronal section, enlargement of the anterior horn of the lateral ventricle, and atrophy of the striatum and frontal lobe with thinner corpus callosum are observed; however, the amygdala and temporal lobe are preserved. (A, B: bar=1 cm). (C) Primary motor cortex showing neuronal loss with astroglial proliferation and spongiform changes in the upper cortical layers. (Bar=500 μm). (D) Caudate nucleus showing marked neuronal loss with gliosis. The arrow indicates a basophilic cytoplasmic inclusion. (Bar=50 μm, C, D: hematoxylin and eosin staining).
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Fig. 4. Cervical spinal cord features. (A) Marked atrophy is observed at C7. The posterior column is relatively preserved while the numbers of other fibers were decreased. (A–C: patient 1) (B) Marked neuronal loss and gliosis of the anterior horn are observed at C7. (C) Marked fiber loss in both the lateral corticospinal tract and spinocerebellar tract (*) is observed at C7. (D) Degeneration in the anterior horn and lateral to the anterior column, particularly in pyramidal tract and preserved posterior column, are observed in the cervical spinal cord. (D–F: patient 2) (E) Marked neuronal loss and gliosis of the cervical anterior horn is observed. (F) Many macrophages and astroglial proliferation are observed in the lateral corticospinal tract. (A, D, F: Klüver–Barrera staining, bar = 0.5 cm, B, E: hematoxylin and eosin staining, C: Bodian staining, B, C, E, F: bar = 50 μm).
(Fig. 4A, C). Nerve fibers of the brainstem were barely observed except for relative preservation in the temporoparietooccipitopontine tract, medial lemniscus, pontocerebellar fibers and middle cerebellar peduncle. Only a few Betz cells were observed and these were atrophic and generalized astroglial proliferation in all layers were observed in the primary motor cortex. Furthermore, the primary motor cortex, primary somatosensory cortex, and frontal and parietal cortices showed neuronal loss with gliosis. In these cortices, the upper cortical layers showed spongiform changes, and the deep cortical layers showed a loss of the radiating myelinated fibers (Fig. 3C). Nerve fibers in the frontal white matter were markedly decreased in number. The following systems were severely affected by the disease (Table 1): the basal ganglia, which were severely affected in the striatum (Fig. 3D), subthalamic nucleus, and substantia nigra, and were mildly affected in the globus pallidus; the cerebellar dentate nucleus and efferent fibers and the red nucleus; the pontine nucleus, the inferior olive, and Purkinje cells. The presence of BIs were difficult to establish in the lower motor neurons, the neurons of the substantia nigra and in the cerebellar dentate nucleus because of their marked neuronal loss (Table 1). The caudate nucleus showed severe degeneration, and many BIs were found there (Fig. 3D). In contrast, BIs were rarely found in the
limbic area and occipital cortex. The BIs were round in shape and compactly or loosely packed (Fig. 5A), sometimes with distinct basophilic rims. The BIs were positive with Bodian staining, while negative with PAS, cresyl violet, LFB, or Gallya–Braak staining.
4.2. Neuropathological findings in patient 2 Limited anatomical regions were available for neuropathological analysis. The spinal cord atrophy was mild (Fig. 4D), and the anterior horn and hypoglossal nucleus showed mild atrophy in spite of marked neuronal loss with astroglial proliferation (Fig. 4E). Nerve fiber loss with numerous macrophages was observed in the corticospinal tract (Fig. 4F) and spinocerebellar tract. The corticospinal tract showed the same features as the internal capsule. However, the primary motor cortex, the hippocampus and the amygdala were not examined. Numerous BIs were observed in extensive areas with slight degeneration (Table 1). The shape of the BIs in patient 2 was similar to that in patient 1, especially in the anterior horn of the spinal cord and the pigmented neurons of the substantia nigra. However, some BIs in the globus pallidus, thalamus, pontine nucleus, inferior olivary nucleus, and dentate nucleus of
Y. Mochizuki et al. / Journal of the Neurological Sciences 323 (2012) 85–92 Table 1 Neuropathological findings.
Motor neurons Primary motor cortex Anterior horn of the spinal cord Hypoglossal nucleus Oculomotor nucleus Frontal cortex Basal ganglia and substantia nigra Caudate nucleus Putamen Globus pallidus Substantia nigra Cerebellum and related area Purkinje cells Cerebellar dentate nucleus Red nucleus Inferior olivary nucleus Pontine nucleus
Patient
1
Patient
2
DEG
BI
NCI
DEG
BI
NCI
++ +++ +++ +++ ++
2 1 − 1 2
2 1 − 1 2
n +++ ++ − +
n 2 1 1 1
n 3 1 1 1
+++ +++ ++ +++
3 2 1 −
3 1 − −
+ − + +
1 1 1 1
2 2 3 3
++ +++ ++ ++ ++
− 1 − − 2
− − − 1 2
+ + + − −
− 2 1 2 1
− 3 1 3 2
DEG: degeneration assessed on the hematoxylin and eosin-, Klüver-Barrera-stained sections. The degeneration was indicated as absent (−), slight (+), mild (++) or severe (+++). BIs: basophilic inclusions. NCI: FUS-immunoreactive (ir) neuronal cytoplasmic inclusion, n: not evaluated (or not examined) The BIs and the FUS pathology was indicated as none (−), rare (1), occasional (2), frequently (3).
patient 2 were irregular in shape. Moreover, in patient 2, some cytoplasmic inclusions showed weak eosinophilia. 4.3. Immunohistochemistry of BIs Both the BIs and the weak eosinophilic inclusions were strongly immunoreactive (ir) for FUS (Fig. 5B). The FUS-ir neuronal cytoplasmic inclusions (NCIs) were more numerous and widespread than NCIs identified with HE, KB, or Bodian staining (Table 1). A few FUS-ir neuronal intranuclear small round inclusions were observed in the cerebral cortex in patient 1 (Fig. 5C) and in the cerebellar dentate nucleus in patient 2. Some FUS-ir granular deposits were observed in the neuronal cytoplasm and neuritis (Fig. 5D). The BIs were slightly positive or negative for ubiquitin, while negative for TDP-43, α-internexin, AT8, and α-synuclein immunoreactivity. Some FUS-ir glial inclusions were observed in the cerebral cortex and the white matter in patient 1. The FUS-ir NCIs of the cerebral cortex in patient 1 appeared mainly in the frontal cortex, including the primary motor cortex and, to some extent, in the superior parietal lobe but rarely in the primary somatosensory cortex, temporal cortex or occipital cortex. There was no immunoreactivity in the granular neurons, while a few pyramidal neurons of the hippocampus were immunoreactive for FUS. 4.4. Electron microscopical findings of BIs The BIs in patient 1 were loose clusters of filamentous structures associated with granules and had no limiting membrane (Fig. 5E). The BIs occasionally contained cytoplasmic organelles, such as mitochondria, but no neurofilaments with side arms or twisted tubules were found in them (Fig. 5F). 5. Discussion All three patients in the family described in this case study developed limb weakness followed by bulbar palsy at a young age with a very rapid progression. In patient 1, the FUS P525L mutation was identified. Therefore, although a gene analysis was not conducted in the other two affected patients, it seems likely that they had the same FUS mutation. This clinical phenotype is similar to that of the already reported juvenile ALS patients (Table 2) [1–4,6,10–16]. Furthermore, similar to patient 1, some reported juvenile ALS patients
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had developmental delays [1,3,6,14], which suggests an early manifestation of the disease. Despite the short disease duration in patient 2 and the juvenile ALS patients (Table 2) [1–4,6,12–16], their lower motor neuron degeneration was severe (Fig. 4D, E), which seems to be reflected in their very rapid progression of paralysis. Without obvious symptoms of upper motor neuron impairment, the corticospinal tract of patient 2 and patient 8 [6] in Table 2 showed very active mobilization of macrophages (Fig. 4F). These findings suggest that the lower motor neuron degeneration developed earlier than the upper motor neuron degeneration, and both of them were very active. Therefore, in patient 1, it is probable that the very severe degeneration of both the lower and upper motor neurons (Fig. 4A–C), including the primary motor cortex (Fig. 3C) came from those very active features in the early stages of the disease. Furthermore, patient 1 showed severe involvement of multiple systems (Table 1). In all of the regions of patient 2, neuronal loss and gliosis were slight, whereas frequent BIs/FUS-ir NCIs appeared (Table 1). In contrast, the limbic system was preserved with rare BIs/FUS-ir NCIs. These findings suggest that the neuronal loss was preceded by the appearance of the BIs/FUS-ir NCIs. In the juvenile ALS patients (Table 2) [1–4,12,14], although various regions besides motor neurons had the BIs/FUS-ir NCIs, these regions did not showed neuronal loss. The disease duration of the juvenile ALS patients was shorter than patients of this case study because the patients of this case study used mechanical ventilation for more than one year. These results might indicate that the initiation of a neuronal loss needs some period of time from the appearance of the BIs/FUS-ir NCIs to occur. Considering the neuroanatomical lesions, the marked fiber loss in the brainstem and spinal cord (Fig. 4A) of patient 1 appeared similar to respirator-assisted long-survival ALS patients, who progressed into a totally locked-in state [8,17–20]. However, their neuroanatomical lesions are various. Although all the patients in these previous studies [8,17–20] showed severe degeneration of the globus pallidus, substantia nigra, and subthalamic nucleus, degeneration of the striatum and the brain atrophy were not present or were mild [8,17–19], except in one patient [20]. This last patient showed the marked frontal lobe atrophy and the multiple system degeneration including the striatum that were also observed in patient 1. However, the patient from the previous study had widespread TDP-43-ir NCI [20]. Therefore, it was suggested that the degeneration of the striatum was correlated with the frontal atrophy of either FUS or TDP-43 pathology. In view of the connections among neurons, the striatum is well known to be the main entry point from the motor-related cortex and connected by the radiating fibers, which come from the deep cortical layers. The loss of radiating fibers in patient 1 corresponded to this feature. The other degenerated lesions in the pontine nucleus and subthalamic nucleus were also connected with the motor-related cortex. Furthermore, in the upper cortical layer, degeneration was contributed to loss of the association fibers, which connected the cerebral cortex. Therefore, the loss of the radiating and associated fibers in the primary motor cortex and the related cortex might induce multiple system involvement, concomitantly with the cerebral white matter atrophy. Regarding the respirator-assisted long-survival ALS with the other mutation in the FUS gene, there has been one ALS patient with the FUS R521C mutation. This patient became quadriplegic with ophthalmoparesis and neuropathologically showed multiple systems degeneration with the BIs/FUS-ir NCI and FUS-ir glial cytoplasmic inclusions [7]. Among the findings of the present case study, the feature of ganglion cell loss of the dorsal root ganglia posterior column degeneration was not observed. Another study has described a patient suggested to have the FUS R521C mutation, with similar findings [21]. Her disease duration was only three years without using mechanical ventilation. Therefore, posterior column degeneration is considered to be a characteristic of ALS with the
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Fig. 5. Basophilic cytoplasmic inclusions (BIs). (A) BIs (arrows) are round (hematoxylin and eosin staining). (B) BIs are strongly immunoreactive (ir) for fused in sarcoma (FUS). (A, B: primary motor cortex of patient 1). (C) FUS-ir neuronal intranuclear inclusions are small and round. (D) FUS-ir deposits are rarely found in neurites. (C, D: parietal cortex of patient 1. A–D: bar = 10 μm). (E) Electron micrograph of a BI (arrows). Note its globular shape with no limiting membrane. (Pons of patient 1, N: nucleus, *BI, bar = 5 μm). (F) At higher magnification, the filamentous structures appeared to be straight or curved with a diameter of range from 15 to 25 nm and had no side arm or constriction. The granules were 20–30 nm in diameter. (Inferior frontal cortex of patient 1, bar = 500 nm, C, D: uranyl acetate and lead citrate staining.)
FUS R521C mutation. According to this feature, it is probable that there are some differences in the disease phenotype between ALS patients with the FUS P525L mutations and those with R521C mutations. Furthermore, the findings of ALS patients with the FUS R521C mutations are of interest because posterior column degeneration is known to be the characteristic of ALS patients with a mutation of the copper/zinc superoxide dismutase (SOD1) gene. A patient with the SOD1 V118L mutation, who progressed into a totally locked-in state, showed posterior column degeneration [19]. Based on the finding that posterior column degeneration is characteristic in ALS patients with either FUS or SOD1 gene mutations, the disease phenotype may have some factors besides gene mutation. Although variability in the morphology and tinctorial characteristics of BIs has been noted [4], the ultrastructural profiles of BIs in the present patients were essentially similar to those of BIs in other diseases that were recently reported to have a FUS-ir pathology [22]. The diseases with BIs and a FUS-ir pathology include juvenile sporadic ALS [2,6,14], adult-onset sporadic ALS [23,24], and generalized
variants of Pick's disease [25], which are now classified as basophilic inclusion body disease subtypes of FTLD-FUS [5,26]. Furthermore, the findings of striatum and frontal lobe atrophy with frequent FUS-ir NCIs [5,22–29] were the same as the findings observed in the present patients. However, the preserved amygdala and hippocampus and no vermiform FUS-ir neuronal intranuclear inclusions, a consistent feature of FTLD-FUS observed in the present patients were different from the findings of FTLD-FUS patients [5,26]. Therefore, ALS with the FUS P525L mutation might have a partially common pathology to FTLD-FUS. In conclusion, we examined three members in a family with a very rapid progressive ALS with the FUS P525L mutation. In contrast to patient 1, who progressed into a totally locked-in state and showed multiple systems degeneration in addition to marked motor neuron degeneration, patient 2 died in the early stage of the disease and showed very active motor neuron degeneration and slight multiple systems degeneration with frequent BIs/FUS-ir NCIs. The clinicopathological findings of these patients indicate that the degeneration
− UE −
− + − − − − [15]
+ UE −
− + − − − − [14]
− UE Dementia, ophthalmoparesis, autonomic symptom
Conflict of interest + Various locations + − − − − [13] [14] + + − + + + [1] + + − + NR NR [3,4]
Acknowledgments
+ + − − − − [6] + + − − − + [3] W: woman, M: man, NIPPV: non−invasive positive pressure ventilation, NR: not recorded, LE: lower exteremities, UE: upper extremities. # Deletion mutation in exon 15 of the FUS gene.
+ + + + + + [2] + + − − − − [6] + + + + + + [3,4] Apearance of FUS-immunoreactive neuronal cytoplasmic inclusions or basophilic inclusions Motor cortex + NR No No No autopsy Lower motor neurons + + autopsy autopsy Basal ganglia + + Pontine nucleus + + Cerebellar dentate nucleus + + Substantia nigra − + Reference Present patients [10] [11]
+ + − + + + [3,4]
+ − UE UE − − − UE − − Hip − NR NR NR − UE NR + NR LE UE Ophthalmoparesis −
The authors have no conflicts of interest to report.
+ + − − − + [12]
+ − UE UE − Autonomic symptom + UE −
No 24 W 7 No 16 W 12
No 25 W 37 * 27 − UE −
No 21 W 4
− NR −
No 12 W 12 No 18 W 6
No 13 W 20 * 4 (NIPPV) + LE −
No 15 M 18
Sporadic juvenile ALS with basophilic cytoplasmic inclusions #
No 18 W 11 No 22 W 10
Yes 16–32 2 W, 2 M b12 in 3, b24 in 1 − UE, Bulbar − Yes 22 NR 6 Yes 35 W 6 Yes 27 W 28 * 14 FUS P525L mutation
Yes 13 W 312 *294
Famiy history Age at onset (years) Gender Duration*respiratory assist (months) Developmental delay Initial symptom Atypical symptoms as ALS
91
spread over multiple systems during ALS with the FUS P525L mutation is caused by not only the appearance of the BIs/FUS-ir NCIs but also the degeneration of the neuronal connection from the primary motor cortex and related areas. The degeneration of the striatum might be considered to be the cause of frontal lobe degeneration in ALS patients.
− + − − − − [16]
No 11 W 12 No 22 W 6 No 19 W 32 *several
− UE −
18 6 5 4 3 2 1 Patient
Table 2 Clinical features and distribution of basophilic cytoplasmic inclusions of juvenile amyotrophic lateral sclerosis.
7
8
9
10
11
12
13
14
15
16
17
19
No 14 M b12
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The authors are grateful to J. Motoki, A. Ishihara, Y. Sato, and others in the laboratory at the Tokyo Metropolitan Neurological Hospital and to K. Suwabe, S. Aoyagi, M. Tano, and E. Kawakami at the Tokyo Metropolitan Institute for Neuroscience for their excellent technical assistance. This study was supported in part by grants from the Japan Society for the Promotion of Science (JSPS) Kakenhi Grant-in-Aid for Scientific Research (B) 22390429 and a grant from the Joint Program for ALS Research, Tokyo Metropolitan Institute of Medical Science (Y. M), and Research on Measures for Intractable Diseases (H23-nanchi-ippan-06 and H23-nanchi-ippan-013), a Grant-in-Aid for Scientific Research (C, 23500435) and a Grantin-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the Ministry of Education, Culture, Sports, Science and Technology (M. T.), and the Japanese Ministry of Education, Science, Sports and Culture, Basic Research (B) #22300177 (K. O.). References [1] Nelson JS, Prensky AL. Sporadic juvenile amyotrophic lateral sclerosis. A clinicopathological study of a case with neuronal cytoplasmic inclusions containing RNA. Arch Neurol 1972;27:300-6. [2] Oda M, Akagawa N, Tabuchi Y, Tanabe H. A sporadic juvenile case of the amyotrophic lateral sclerosis with neuronal intracytoplasmic inclusions. Acta Neuropathol 1978;44:211-6. [3] Bäumer D, Hilton D, Paine SML, Turner MR, Lowe J, Talbot K, et al. Juvenile ALS with basophilic inclusions is a FUS proteinopathy with FUS mutations. Neurology 2010;75:611-8. [4] Mackenzie IRA, Ansorge O, Strong M, Bilbao J, Zinman L, Ang LC, et al. Pathological heterogeneity in amyotrophic lateral sclerosis with FUS mutations: two distinct patterns correlating with disease severity and mutation. Acta Neuropathol 2011;122:87-98. [5] Mackenzie IRA, Munoz DG, Kusaka H, Yokota O, Ishihara K, Roeber S, et al. Distinct pathological subtypes of FTLD-FUS. Acta Neuropathol 2011;121:207-18. [6] Huang EJ, Zhang J, Geser F, Trojanowski JQ, Strober JB, Dickson DW, et al. Extensive FUS-immunoreactive pathology in juvenile amyotrophic lateral sclerosis with basophilic inclusions. Brain Pathol 2010;20:1069-76. [7] Tateishi T, Hokonohara T, Yamasaki R, Miura S, Kikuchi H, Iwaki A, et al. Multiple system degeneration with basophilic inclusions in Japanese ALS patients with FUS mutation. Acta Neuropathol 2010;119:355-64. [8] Hayashi H, Kato S. Total manifestations of amyotrophic lateral sclerosis. ALS in the totally locked-in state. J Neurol Sci 1989;93:19-35. [9] Hayashi H, Oppenheimer EA. ALS patients on TPPV: totally locked-in state,159 neurologic findings and ethical implications. Neurology 2003;61:135-7. [10] Kwiatkowski Jr TJ, Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 2009;323:1205-8. [11] Chiò A, Restagno G, Brunetti M, Ossola I, Calvo A, Mora G, et al. Two Italian kindreds with familial amyotrophic lateral sclerosis due to FUS mutation. Neurobiol Aging 2009;30:1272-5. [12] Aizawa H, Kimura T, Hashimoto K, Yahara O, Okamoto K, Kikuchi K. Basophilic cytoplasmic inclusions in a case of sporadic juvenile amyotrophic lateral sclerosis. J Neurol Sci 2000;176:109-13. [13] Berry RG, Chambers RA, Duckett S. Clinicopathological study of juvenile ALS. Neurology 1969;19:312. [14] Matsumoto S, Kusaka H, Murakami N, Hashizume Y, Okazaki H, Hirano A. Basophilic inclusions in sporadic juvenile amyotrophic lateral sclerosis: an immunocytochemical and ultrastructural study. Acta Neuropathol 1992;83:579-83. [15] Tsujihata M, Taguchi H, Oku Y, Takamori M, Terao H. A sporadic case of juvenile ALS. Rinsho Shinkeigaku 1978;18:82-8 (in Japanese with English abstract). [16] Wohlfart G, Swank RL. Pathology of ALS. Fiber analysis of the ventral roots and pyramidal tracts of the spinal cord. Arch Neurol Psychiatry 1941;46:783-99.
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